Interaction of bim with trim2, an e3 ubiquitin ligase

Abstract

Methods, compositions, and cells for drug screening based on interaction between (1) a Bim polypeptide and/or an E2 polypeptide and (2) a TRIM2 polypeptide, which is identical to or corresponds to a Bim-selective E3 ubiquitin ligase. Methods and compositions for treating cancer based on levels of Bim protein, TRIM2 protein, and active MAPK protein are also provided.

1 . A method of drug screening, comprising:
 forming an assay mixture including a Bim polypeptide, a TRIM2 polypeptide, and one or more test compounds;   detecting interaction of the Bim and TRIM2 polypeptides with each other; and   determining whether the one or more test compounds affect the interaction.   
   
   
       2 . The method of  claim 1 , further comprising a step of selecting at least one test compound as a candidate drug for treatment of a disease characterized by an abnormal amount of cell death, based at least in part on the step of determining. 
   
   
       3 . The method of  claim 1 , wherein the step of selecting selects at least one test compound that inhibits the interaction, such that the at least one test compound is a candidate drug for treating cancer by increasing cell death. 
   
   
       4 . The method of  claim 1 , wherein the step of selecting selects at least one test compound that enhances the interaction, such that the at least one test compound is a candidate drug for reducing cell death associated with neurological disease. 
   
   
       5 . The method of  claim 1 , wherein at least one of the Bim and TRIM2 polypeptides has a luminescent tag, and wherein the step of detecting includes a step of optically detecting a signal that indicates an amount of interaction of the polypeptides. 
   
   
       6 . The method of  claim 5 , wherein the step of optically detecting a signal includes a step of measuring fluorescence resonance energy transfer or fluorescence polarization. 
   
   
       7 . The method of  claim 5 , wherein the Bim and TRIM2 polypeptides include a respective Bim portion and TRIM2 portion each conjugated to a distinct fluorescent polypeptide. 
   
   
       8 . The method of  claim 1 , further comprising a step of contacting one or both of the Bim and TRIM2 polypeptides with an antibody that recognizes only one of the polypeptides. 
   
   
       9 . The method of  claim 8 , wherein at least one of the Bim and TRIM2 polypeptides includes an epitope tag. 
   
   
       10 . The method of  claim 1 , further comprising a step of phosphorylating the Bim polypeptide before the step of detecting interaction. 
   
   
       11 . The method of  claim 10 , further comprising a step of contacting the Bim polypeptide with a MAP kinase. 
   
   
       12 . The method of  claim 1 , wherein the step of forming an assay mixture includes a step of introducing an inhibitor of MAPK kinase. 
   
   
       13 . The method of  claim 1 , wherein the step of forming an assay mixture is repeated with different test compounds in distinct wells of a microplate. 
   
   
       14 . The method of  claim 1 , wherein the step of detecting interaction is repeated without the one or more test compounds in the assay mixture, and wherein the step of determining is based on a comparison of the interaction detected with and without a test compound. 
   
   
       15 . The method of  claim 1 , wherein the one or more test compounds include a polypeptide identical or corresponding to only a fragment of Bim. 
   
   
       16 . The method of  claim 1 , wherein the assay mixture includes biological cells that express the Bim and TRIM2 polypeptides. 
   
   
       17 . A composition for drug screening, comprising:
 an engineered Bim polypeptide; and   an engineered TRIM2 polypeptide that interacts with the Bim polypeptide.   
   
   
       18 . The composition of  claim 17 , wherein the Bim and TRIM2 polypeptides include a respective Bim portion and TRIM2 portion each conjugated to a distinct fluorescent polypeptide, and wherein the fluorescent polypeptides are configured to permit interaction of the Bim and TRIM2 polypeptides to be detected by fluorescence resonance energy transfer. 
   
   
       19 . A method of treating cancer, comprising:
 testing one or more samples from a cancer patient for levels of TRIM2 protein, Bim protein, and active MAP kinase protein; and   treating the cancer patient with at least one drug that inhibits degradation of Bim protein if the level of Bim protein is below a first threshold, the level of TRIM2 protein is above a second threshold, and the level of active MAP kinase protein is above a third threshold.   
   
   
       20 . The method of  claim 19 , wherein the at least one drug includes a MAPK kinase inhibitor, a proteasome inhibitor, or both.